Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.

Two DNA segments, dnrR1 and dnrR2, from the Streptomyces peucetius ATCC 29050 genome were identified by their ability to stimulate secondary metabolite production and resistance. When introduced into the wild-type ATCC 29050 strain, the 2.0-kb dnrR, segment caused a 10-fold overproduction of e-rhodomycinone, a key intermediate of daunorubicin biosynthesis, whereas the 1.9-kb dnrR2 segment increased production of both r-rhodomycinone and daunorubicin 10-and 2-fold, respectively. In addition, the dnrR2 segment restored high-level daunorubicin resistance to strain H6101, a daunorubicin-sensitive mutant of S. peucetius subsp. Knowledge about the genetics of secondary metabolism in Streptomyces spp. has grown rapidly since the reports 7 years ago that described the cloning of an apparent O-methyltransferase gene (11) and the entire cluster of actinorhodin production genes (33) from Streptomyces coelicolor. Although our understanding of the molecular biology of secondary metabolism has grown considerably in the ensuing years, one question stands out among the topics of current interest. How is the expression of antibiotic production genes regulated, both by genes that are linked to the structural and, commonly, self-resistance genes and by unlinked loci that could influence secondary metabolism indirectly? Information about this question is fundamentally important to understanding the genetics of temporally regulated processes in these filamentous soil bacteria (19) and should lead to ways to construct recombinant strains that overproduce valuable microbial metabolites (6).Insight into regulation by closely linked genes has been obtained from studies of mutations within the cluster of production genes that interfere with the functions of most or all of the other genes in this region. For instance, S. coelicolor actII strains do not produce actinorhodin and do not cosynthesize it with mutants that accumulate intermediates of actinorhodin biosynthesis (45), suggesting that the actII locus has a central role in actinorhodin production different from the role played by the structural and resistance genes (34). In contrast, studies of pleiotropic muta-* Corresponding author. tions have provided information about regulation by unlinked genes, since these mutations affect antibiotic production as well as other characteristics that are known to be developmentally regulated (5, 19), like the formation of aerial mycelia and spores. S. coelicolor bldA strains, for example, exhibit defects in the formation of aerial mycelia and antibiotics, leading to the belief that these properties are mediated by the bldA product in the wild-type strain via a novel type of translational control (31). The S. coelicolor afsR gene seems to bridge the actions of actII and bldA, since afsR can modulate the function of both the act and red clusters (and possibly A-factor production [22,24]) but has no proven role in development (25).The properties of the S. coelicolor actII-orf4 gene, recently described by , and the S. coelicolor redD-orfl gene (37)...

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Genes for the biosynthesis of daunorubicin (daunomycin) and doxorubicin (adriamycin), important antitumor drugs, were cloned from Streptomycespeucetius (the daunorubicin producer) and S. peucetius subsp. caesius (the doxorubicin producer) by use of the actIltemla and actII polyketide synthase gene probes. Restriction Daunorubicin (daunomycin) and doxorubicin (adriamycin) are commercially important antibiotics with potent antitumor activity. Daunorubicin, first isolated in 1963 from Streptomyces peucetius (6, 7), was subsequently found in a number of other Streptomyces spp. (27). Doxorubicin was isolated in 1969 from S. peucetius subsp. caesius, a mutant of the wild-type strain (2), and has important clinical applications in cancer chemotherapy (1), even though both doxorubicin and daunorubicin cause cardiotoxic side effects that are dose limiting and irreversible. The production costs of these antibiotics are high because of low titers and formation of a complex mixture of products by the producing bacteria. Therefore, a genetic study of the biosynthesis of daunorubicin and doxorubicin was undertaken in our laboratory to elucidate the organization and regulation of the biosynthetic genes, with the hope that it may also lead to overproducing strains or strains with a simpler spectrum of secondary metabolites. This work has been facilitated by the recognition that the early genes involved in polyketide biosynthesis by other streptomycetes, such as the S. coelicolor actI and actIlI genes (9, 17) and the S. glaucescens tcmIa genes (20), hybridize to the analogous genes in other polyketide producers (16) and by the fact that antibiotic genes have been found to be clustered in all of the examples studied (14).A previous report (24) from this laboratory demonstrated that the daunorubicin producer S. peucetius (ATCC 29050) contains five nonoverlapping regions of DNA that hybridized to the actIl/tcmIa or actIII probe. The properties of S. peucetius and S. lividans strains transformed with clones from four of these regions supported the belief that many of the daunorubicin production (dnr) genes resided in region IV but also raised the possibility that bona fide dnr genes or genes that influence daunorubicin production and self-resistance were present in the three other regions (24). The latter idea is best tested by examining the effects of deletions in * Corresponding author.each of these four regions on daunorubicin production and resistance.Since our preliminary evidence (24) indicated that some portion of region II had been deleted from S. peucetius subsp. caesius (ATCC 27952), we made a detailed study of the properties of cosmid clones from three nonoverlapping regions of DNA in this strain. The effects of these clones and additional clones from the wild-type 29050 strain in homologous and heterologous hosts confirm that the genes required for formation of e-rhodomycinone, a key intermediate of daunorubicin biosynthesis, are present in region IV. In addition, we demonstrated that this region contains two daunorubi...